HPLC–MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEMss cells

A sensitive and specific method using high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC–MS/MS analysis. ¹³C₅-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245 → 113 and 250 → 133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01–10 μg/mL with a limit of quantitation of 0.01 μg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 μg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM_ss cells cultured with RBV.     

Determination of dexmedetomidine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: Application to a pharmacokinetic study

A rapid, sensitive and selective high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method (HPLC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of dexmedetomidine (DMED) in human plasma. Dexmedetomidine and the internal standard (ondansetron) were extracted in a single step with diethyl-ether from 1.0 mL of alkalinized plasma. The mobile phase was a mixture of acetonitrile and 0.5% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]⁺ ions m/z 201.0 → 95.1 for DMED and m/z 294.1 → 170.1 for the IS. The assay exhibited a linear dynamic range of 5–5000 pg mL⁻¹ with the correlation coefficient above 0.9995. The lower limit of quantification (LLOQ) was 5 pg mL⁻¹ with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated HPLC-MS/MS method has been successfully applied to study the pharmacokinetics of three level doses of DMED in Chinese healthy volunteers.     

Quantification of CKD-501, lobeglitazone, in rat plasma using a liquid-chromatography/tandem mass spectrometry method and its applications to pharmacokinetic studies

CKD-501 (i.e., lobeglitazone), a potent agonist for both PPARα/γ, is a new drug that has potential clinical applications in the management of type-2 diabetes. The objective of this study was to develop a rapid and sensitive method for the determination of CKD-501 in rat plasma and to assess the applicability of the assay to pharmacokinetic studies. Rat plasma samples were processed using a fast flow protein precipitation (FF-PPT) method and then introduced onto an LC–MS/MS system for quantification. The analyte and rosiglitazone, an internal standard, were analyzed by multiple reactions monitoring (MRM) at m/z transitions of 482.0 → 258.0 for CKD-501 and 358.0 → 135.0 for the internal standard. The lower limit of quantification (LLOQ) was determined at 50 ng/mL, with an acceptable linearity in the range from 50 to 10,000 ng/mL (R > 0.999). Validation parameters such as accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the acceptance criteria of the assay validation guidelines, indicating that the assay is applicable to estimating the concentration in the range studied. The concentration of CKD-501 was readily quantifiable in plasma samples up to 24 h post-dose in rats that had received an oral dose of 1 mg/kg. These observations suggest, therefore, that the validated assay can be used in pharmacokinetic studies of CKD-501 in small animals such as the rat.     

Simultaneous determination of nine major active compounds in Dracocephalum rupestre by HPLC

A new method was developed for the simultaneous determination of nine major constituents in Dracocephalum rupestre, including 5,7-dihydroxychromone (1), eriodictyol-7-O-β-d-glucoside (2), luteolin-7-O-β-d-glucoside (3), naringenin-7-O-β-d-glucoside (4), apigenin-7-O-β-d-glucoside (5), eriodictyol (6), luteolin (7), naringenin (8) and apigenin (9). The quantitative determination was conducted by reversed phase high-performance liquid chromatography with photodiode array detector (LC–PDA). Separation was performed on an Agilent Eclipse XDB-C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.5% aqueous acetic acid. The components were identified by retention time, ultraviolet (UV) spectra and quantified by LC–PDA at 260 nm. All calibration curves showed good linearity (r² > 0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.0%. The recoveries were between 95.15 and 104.45%. The limits of detection (LOD) ranged from 0.002 to 0.422 μg/ml and limits of quantification (LOQ) ranged from 0.005 to 1.208 μg/ml, respectively. The identity of the peaks was further confirmed by high-performance liquid chromatography with triple-quadrupole mass spectrometry system coupled with electrospray ionization (ESI) interface. The developed method was applied to the determination of nine constituents in 14 samples of D. rupestre collected at various harvesting times. Most compounds accumulated at much higher amounts in about June–July. The satisfactory results indicated that the developed method was readily utilized as a quality control method for D. rupestre.     

LC-MS/MS method for determination of hederacolchiside E, a neuroactive saponin from Pulsatilla koreana extract in rat plasma for pharmacokinetic study

A simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was applied to pharmacokinetic study of a neuroactive oleanolic-glycoside saponin, hederacolchiside E from SK-PC-B70M, a standardized extract of Pulsatilla koreana in rat. Rat plasma samples were pretreated by protein precipitation with acetonitrile, eluted from C₁₈ column, and analyzed using electrospray ionization (ESI)-MS/MS in negative ion mode. Digoxin was used as an internal standard. The standard curves were linear (r > 0.997) over the concentration ranges of 2–500 ng/mL. The intra- and inter-day precisions were measured to be below 9% and accuracy between 90 and 111% for all quality control samples at 2, 20, 100, and 500 ng/mL (n = 5). The lower limits of quantification (LLOQ) for hederacolchiside E was 2 ng/mL and the limit of detection (LOD) 0.5 ng/mL using 20 μL of plasma sample. Subsequently, hederacolchiside E was determined in rat plasma samples after oral administration of SK-PC-B70M. The mean maximum plasma concentrations of hederacolchiside E were 0.07, 0.13, and 0.36 μg/mL and the mean areas under the plasma concentration versus time curve 0.56, 1.27, and 6.46 μg h/mL at doses of 100, 200, and 400 mg/kg, respectively, which indicated non-linear pharmacokinetic pattern. In conclusion, this method was successfully applied to the pharmacokinetic study of hederacolchiside E after an oral administration of SK-PC-B70M to rats.     

Antihypertensive and vasorelaxant activities of Laelia autumnalis are mainly through calcium channel blockade

The aim of the present study was to evaluate the possible mechanism of the vasorelaxant action of methanol extract from Laelia autumnalis (MELa) in isolated rat aortic rings, and to establish its antihypertensive activity in vivo. MELa (0.15→50 µg/mL) induced relaxation in aortic rings pre-contracted with KCl (80 mM), showing an IC₅₀ value of 34.61 ± 1.41 µg/mL and E_max value of 85.0 ± 4.38% (in endothelium-intact rings) and an IC₅₀ value of 45.11 ± 4.17 µg/mL and E_max value of 80.0 ± 12.1% (in endothelium-denuded rings). Serotonin (5-HT, 1 × 10^− 4 M) provoked sustained contraction, which was markedly inhibited by MELa (0.15→50 µg/mL) in a concentration-dependent and endothelium-independent manner. Pretreatment with MELa (15, 46, 150, 300 and 1500 µg/mL) also inhibited contractile responses to norepinephrine (NE 1 × 10^− 11 M to 1 × 10^− 5.5 M). In endothelium-denuded rings, the vasorelaxant effect of MELa was reduced partially by ODQ (1 µM), but not by tetraethylammonium (5 µM), glibenclamide (10 µM), and 2-aminopyridine (100 µM). The extract also reduced NE-induced transient contraction in Ca²⁺-free solution, and inhibited contraction induced by increasing external calcium in Ca²⁺-free medium plus high KCl (80 mM). The antihypertensive effect of MELa was determined in spontaneously hypertensive rats (SHR). A single oral administration of the extract (100 mg/kg) exhibited a significant decrease in systolic and diastolic blood pressure and heart rate (p < 0.05) in SHR rats. Our results suggest that MELa induces relaxation in rat aortic rings through an endothelium-independent pathway, involving blockade of Ca²⁺ channels and a possible cGMP enhanced concentrations and also causes an antihypertensive effect.     

Determination of ranolazine in human plasma by LC-MS/MS and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of ranolazine in human plasma. The analytical method consists in the precipitation of plasma sample with methanol, followed by the determination of ranolazine by an LC–MS/MS. The analyte was separated on a Peerless Cyano column (33 mm × 4.6 mm, 3 μm) an isocratic mobile phase of methanol–water containing formic acid (1.0%, v/v) (65:35, v/v) at a flow rate of 1.0 ml/min. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard (IS). The MS/MS detection was made by monitoring the fragmentation of m/z 428.20 → 279.50 for ranolazine and m/z 448.30 → 285.20 for internal standard on a triple quadrupole mass spectrometer. The method was validated over the concentration range of 5–2000 ng/ml for ranolazine in human plasma with correlation coefficient of 0.9937 (S.D.: ±0.00367, range: 0.9895–0.9963). The accuracy and precision values obtained from six different sets of quality control samples analyzed in separate occasions ranged from 94.53 to 117.86 and 0.14% to 4.56%, respectively. Mean extraction recovery was 82.36–94.25% for three quality control (QC) samples and 88.37% for IS. Plasma samples were stable for three freeze–thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at −20 °C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 °C). The developed method was successfully applied for analyzing ranolazine in plasma samples for a bioequivalence study with 12 healthy volunteers.     

Intracerebral microdialysis coupled to LC–MS/MS for the determination tramadol and its major pharmacologically active metabolite O‐desmethyltramadol in rat brain microdialysates

A rapid and sensitive method involving liquid chromatography electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) coupled to an intracerebral microdialysis technique was developed for the determination and pharmacokinetic investigation of tramadol and its major active metabolite O ‐desmethyltramadol (ODT) in rat brain. The microdialysis samples were separated on a C18 column and eluted with a mobile phase of acetonitrile‐water‐formic acid (50:50:0.1; v/v/v ) at a flow rate of 0.3 mL/min. The ESI‐MS/MS spectra were performed in electrospray positive ion mode, and the analytes were detected by multiple reaction monitoring (MRM) of the transitions m/z [M + H]⁺ 264.3 → 58.2 for tramadol, m/z [M + H]⁺ 250.3 → 58.3 for ODT, and m/z [M + H]⁺ 379.4 → 264.0 for ambroxol (internal standard; IS). The total run time was 4.0 min. A lower limit of quantitation (LLOQ) was achieved as 1 ng/mL for tramadol and 0.5 ng/mL for ODT, with excellent linearity over a concentration range of 1 ~ 500 ng/mL (r  > 0.99) for tramadol and 0.5 ~ 50 ng/mL for ODT (r  > 0.99), respectively. The proposed method was successfully applied to the pharmacokinetic studies of tramadol and ODT in rat brain. Copyright © 2017 John Wiley & Sons, Ltd.     

Determination of lansoprazole in human plasma by rapid resolution liquid chromatography–electrospray tandem mass spectrometry: Application to a bioequivalence study on Chinese volunteers

A simple, sensitive and rapid LC/MS/MS method was developed for the quantification of lansoprazole in human plasma. After a simple sample preparation procedure by one-step protein precipitation with acetonitrile, lansoprazole and the internal standard bicalutamide were chromatographed on a Zorbax SB-C₁₈ (3.0 mm × 150 mm, 3.5 μm, Agilent) column with the mobile phase consisted of methanol–water (70:30, v/v, containing 5 mM ammonium formate, pH was adjusted to 7.85 by 1% ammonia solution). Detection was performed on a triple quadrupole tandem mass spectrometry by multiple reaction monitoring (MRM) mode via negative eletrospray ionization source (ESI⁻). The lower limit of quantification was 5.5 ng/mL, and the assay exhibited a linear range of 5.5–2200.0 ng/mL. The validated method was successfully applied to investigate the bioequivalence between two kinds of preparation (test vs. reference product) in twenty-eight healthy male Chinese volunteers.     

Development and validation of a high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemether and its active metabolite dihydroartemisinin in human plasma

To study the pharmacokinetic profile of artemether in children and in the context of antiviral drugs for HIV infected patients co-infected with malaria, an LC-MS/MS method was developed and validated to simultaneously determine artemether and its metabolite dihydroartemisinin in human plasma. Using artemisinin as the internal standard, 0.5 mL samples were processed with solid phase extraction (Waters Oasis^® HLB column), the elutes were directly injected onto a C₁₈ LC column (Waters, Symmetry^®, 150 mm × 4.6 mm, 5 μm). Mass detection utilized ESI⁺ as the ionization mode and MRM as the quantitation mode. In respect to the low ionization capacity of artemether, ammonium formate was added to the LC mobile phase to facilitate ionization (M+NH₄⁺). The calibration range was 2–200 ng/mL. The recovery was 73–81% for artemether and 90–99% for dihydroartemisinin. The validated method was applied to analysis of clinical samples with results in good agreement with an existing method.     

Development of a LC–MS/MS-based method for determining metolazone concentrations in human plasma: Application to a pharmacokinetic study

In this study, a method was developed and validated for the quantification of metolazone in human plasma samples. This method involves high-performance liquid chromatography coupled with tandem mass spectrometry and is more sensitive, selective and rapid than currently available methods. Chromatography was performed using a Phenomenex^® Luna C18 column (100 mm × 2.0 mm, 5 μm, 100 Å) with an isocratic mobile phase of 0.1% formic acid:acetonitrile (40:60, v/v) and zaleplon as an internal standard. The drug and internal standard were extracted by liquid–liquid extraction and analyzed by mass spectrometry in the multiple reaction monitoring mode by using m/z values of 366.20/259.10 for metolazone and 306.20/235.60 for zaleplon. The calibration curve was linear over metolazone concentrations ranging from 0.02 ng/mL to 15 ng/mL. The lower limit of quantification was 0.02 ng/mL. Intra- and inter-assay precisions were 0.9–4.8% and 4.2–6.3%, respectively. The intra- and inter-assay accuracies in quantifying metolazone were 97.5–102.3% and 99.2–104.0%, respectively. Metolazone and zaleplon were eluted within 3.6 minutes, and the retention time was 1.75 minutes for metolazone and zaleplon. The validated method was successfully applied to a pharmacokinetic study of metolazone in human plasma.     

Validated bioanalytical method for the quantification of RGB-286638, a novel multi-targeted protein kinase inhibitor, in human plasma and urine by liquid chromatography/tandem triple-quadrupole mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitative determination of RBG-286638, a novel multi-targeted protein kinase inhibitor, in 200 μl aliquots of human potassium EDTA plasma with deuterated RGB-286638 as internal standard. The sample extraction and cleaning-up involved a simple liquid–liquid extraction with 100 μl aliquots of acetonitrile and 1 ml aliquots of n-butylchloride. Urine was accurately 5- and 10-fold diluted in blank plasma prior to extraction. Chromatographic separations were achieved on a reversed phase C₁₈ column eluted at a flow-rate of 0.250 ml/min on a gradient of 0.2 mM ammonium formate and acetonitrile both acidified with 0.1% formic acid. The overall cycle time of the method was 7 min, with RGB-286638 eluting at 1.9 min. The multiple reaction monitoring transitions were set at 546 > 402 (m/z), and 549 > 402 (m/z) for RGB-286638 and the internal standard, respectively. The calibration curves were linear over the range of 2.00 to 1000 ng/ml with the lower limit of quantitation validated at 2.00 ng/ml. The within-run and between-run precisions were within 7.90%, while the accuracy ranged from 92.2% to 99.7%. The method was successfully applied to samples derived from a clinical study.     

The disposition of biphenylacetic acid following topical application

Summary Plasma and synovial fluid concentrations of biphenylacetic acid were determined following application of 3 g of 3% biphenylacetic acid gel to one knee of patients suffering from rheumatoid arthritis.The mean peak plasma concentration was 34 ng/ml. Synovial fluid concentrations tended to follow plasma concentrations but at a somewhat lower level, the mean peak synovial fluid concentration was 21 ng/ml. The average ratio of synovial fluid AUC (0–24 h) to plasma AUC (0–24 h) was 0.58, r=0.97.Where patients had bilateral effusions, the concentration in the ipsilateral knee at each time point examined was not significantly different to that in the contralateral knee, suggesting that absorption was initially into the plasma and subsequently into the synovium.     

Sensitive determination of 20(S)-protopanaxadiol in rat plasma using HPLC–APCI-MS: Application of pharmacokinetic study in rats

20(S)-Protopanaxadiol (PPD), the main metabolite of protopanoxadiol type ginsenosides (e.g. Rg3 and Rh2), is a very promising anti-cancer drug candidate. To evaluate the pharmacokinetic property of PPD, we reported a reliable, sensitive and simple method utilizing liquid chromatography (HPLC)-atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) to determine PPD. PPD and the internal standard, panoxadiol (PD) were extracted from plasma with acetic ether, separated on a C18 reverse column, and then analyzed by APCI-MS. Targeting fragment ion at m/z 425 for both PPD and PD was monitored in selected-ion monitoring (SIM) mode. PPD can be quantitatively determined at the concentration as low as 1 ng/mL using 200 μL plasma. And the sensitive method showed excellent linearity over a range from 1 to 1000 ng/mL, high recovery, accuracy and precision at the concentrations of 2.5, 100.0 and 1000.0 ng/mL, respectively. The method was successfully applied to pharmacokinetic study of PPD in rats. Pharmacokinetic parameters were calculated and absolute bioavailability of PPD was 36.8 ± 12.4%, at least ten times higher than that of Rg3 and Rh2, indicating its good absorption in gastrointestinal tract. It was further suggested that PPD be a promising anti-cancer candidate and probably responsible for the observed pharmacological activity of Rg3 and Rh2.     

A sensitive LC–MS/MS method to quantify loureirin B in rat plasma with application to preclinical pharmacokinetic studies

A novel method for the quantification of loureirin B in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) was developed. Loureirin B and internal standard (buspirone) were extracted by liquid–liquid extraction and separated on a Agilent XDB C₁₈ column (50 mm × 4.6 mm, 5 μm). As mobile phase a binary mixture of methanol (containing 0.1% formic acid)–water (containing 0.1% formic acid) was delivered by a Shimadzu LC-20AD pump in gradient mode at a flow rate of 0.4 ml/min in a run time of 5.0 min. The detector was a Q-trap™ mass spectrometer with an electrospray ionization (ESI) interface operating in the multiple reaction monitoring (MRM) mode. The calibration curve of loureirin B in plasma showed good linearity over the concentration range of 0.08–100 ng/ml. The limit of detection and limit of quantification were 0.03 ng/ml and 0.08 ng/ml, respectively. Intra- and inter-day precisions (as relative standard deviation) in all samples were both within 15%. The validated method was successfully applied to a preliminary pharmacokinetic study of loureirin B in rats. After oral administration of 16 g/kg longxuejie to rats, the main pharmacokinetic parameters t_max, C_max, t_1/2, K_e and AUC_0–T were 0.8 h, 7.99 μg/l, 1.94 l h, 0.365/h, and 22.21 μg h/l, respectively.     

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(d,l-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents—stearylamine and polyethylenimine—in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague–Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (−6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28–42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels—interferon γ and interleukin-2—were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(d,l-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.     

Detection of Δ9 THC in oral fluid following vaporized cannabis with varied cannabidiol (CBD) content: An evaluation of two point‐of‐collection testing devices

Point‐of‐collection testing (POCT) for Δ⁹‐tetrahydrocannabinol (THC) in oral fluid is increasingly used to detect driving under the influence of cannabis (DUIC). However, previous studies have questioned the reliability and accuracy of two commonly used POCT devices, the Securetec DrugWipe^® 5 s (DW5s) and Dräger DrugTest^® 5000 (DT5000). In the current placebo controlled, double‐blind, crossover study we used liquid chromatography‐tandem mass spectrometry (LC–MS/MS) to accurately quantify cannabinoid concentrations in the oral fluid of 14 participants at various timepoints (10, 60, 120, and 180 minutes) following vaporization of 125 mg of THC‐dominant (11% THC; <1% CBD), THC/CBD equivalent (11% THC; 11% CBD) and placebo (<1% THC; <1% CBD) cannabis. At each timepoint, oral fluid was also screened using the DW5s (10 ng/mL THC cut‐off) and DT5000 (10 ng/mL THC cut‐off). LC–MS/MS analysis showed peak oral fluid THC concentrations at the 10 minute timepoint with a rapid decline thereafter. This trajectory did not differ with THC dominant and THC/CBD equivalent cannabis. With a 10 ng/mL confirmatory cut‐off, 5% of DW5s test results were false positives and 16% false negatives. For the DT5000, 10% of test results were false positives and 9% false negatives. Neither the DW5s nor the DT5000 demonstrated the recommended >80% sensitivity, specificity and accuracy. Accuracy was lowest at 60 minutes, when THC concentrations were often close to the screening cut‐off (10 ng/mL). POCT devices can be useful tools in detecting recent cannabis use; however, limitations should be noted, and confirmatory LC–MS/MS quantification of results is strongly advisable.     

Effect of a metabotropic glutamate receptor 5 antagonist, MPEP, on the nociceptive response induced by intrathecal injection of excitatory aminoacids, substance P, bradykinin or cytokines in mice

Metabotropic glutamate receptors (mGluRs) are expressed abundantly in the spinal cord and have been shown to play important roles in the modulation of nociceptive transmission and plasticity. In this study, the involvement of metabotropic glutamate receptor 5 (mGluR₅) in the nociceptive response induced by intrathecal injection (i.t.) of excitatory aminoacids, substance P (SP), bradykinin (BK) and cytokines in mice was demonstrated. The administration of 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 10–50 nmol/site, i.t.) caused a significant inhibition in the nociceptive response induced by glutamate and trans-ACPD with maximal inhibitory effects of 36 ± 7% and 56 ± 5%, respectively. MPEP completely failed to affect the nociception induced by α-amino-3-hydroxy-5-mehtyl-4-isoxazolepropionic acid (AMPA; 135 pmol/site), kainate (110 pmol/site) and N-methyl-d-aspartate (NMDA; 450 pmol/site). MPEP also reduced the nociceptive response induced by SP (135 ng/site, i.t.), BK (0.1 µg/site), tumor necrosis factor-alpha (TNF-α; 0.1 pg/site) and interleukin-1beta (IL-1β; 1 pg/site) with maximal inhibitions of 29 ± 5%, 37 ± 5%, 83 ± 3% and 88 ± 1%, respectively. Together, these results indicate the involvement of mGluRs, more specifically of subtype-5, in the nociceptive response induced by i.t. injection of excitatory aminoacids, SP, BK and cytokines in mice.